Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics for the targeted delivery of highly potent cytotoxic drugs to tumor cells to improve bioactivity while minimizing side effects. ADCs are composed of both small and large molecules and therefore have complex molecular structures. In vivo biotransformations may further increase the complexity of ADCs, representing a unique challenge for bioanalytical assays. Quadrupole-time-of-flight mass spectrometry (Q-TOF MS) with electrospray ionization has been widely used for characterization of intact ADCs. However, interpretation of ADC biotransformations with small mass changes, for the intact molecule, remains a limitation due to the insufficient mass resolution and accuracy of Q-TOF MS. Here, we have investigated in vivo biotransformations of multiple site-specific THIOMAB antibody-drug conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass (HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS enabled more comprehensive identification of ADC biotransformations. It was particularly beneficial for characterizing ADC modifications with small mass changes such as partial drug loss and hydrolysis. This strategy has significantly enhanced our capability to elucidate ADC biotransformations and help understand ADC efficacy and safety in vivo.

The tubulysins are promising anticancer cytotoxic agents due to the clinical validation of their mechanism of action (microtubule inhibition) and their particular activity against multidrug-resistant tumor cells. Yet their high potency and subsequent systemic toxicity make them prime candidates for targeted therapy, particularly in the form of antibody-drug conjugates (ADCs). Here we report a strategy to prepare stable and bioreversible conjugates of tubulysins to antibodies without loss of activity. A peptide trigger along with a quaternary ammonium salt linker connection to the tertiary amine of tubulysin provided ADCs that were potent . However, we observed metabolism of a critical acetate ester of the drug, resulting in diminished conjugate activity. We were able to circumvent this metabolic liability with the judicious choice of a propyl ether replacement. This modified tubulysin ADC was stable and effective against multidrug-resistant lymphoma cell lines and tumors.

Background Complex lower limb reconstruction due to severe trauma remains a challenge for reconstructive surgeons. Here, we introduce a modified donor blood flow-preserved cross-leg anterolateral thigh flap procedure and evaluate its clinical efficacy. Methods Between January 2013 and December 2019, 22 patients (range 10 to 64 years old) with unilateral lower limb injury underwent modified donor blood flow-preserved cross-leg anterolateral thigh flap procedures. Among them, 16 cases were traffic accidents, 5 cases were persistent ulcers, and 1 case was a degloving injury. The arterial pedicle of the flap was prepared in a Y-shaped fashion and microanastomosed to the posterior tibial artery of intact leg in a flow-through style. A split-thickness skin graft was applied to wrap the vascular pedicle after anastomosis. The flap was designed in a single or bilobed fashion according to the shape of the tissue defect. The operation time, the intraoperative blood loss and the length of hospital stays were recorded. The vascular pedicle was divided 4 weeks after anastomosis. Doppler ultrasound was performed to evaluate the blood flow of the donor posterior tibial artery during postoperative follow-up. Results All 22 flaps survived. The tissue defects ranged from 12 × 6 to 21 × 18 cm2. The flap sizes ranged from 14 × 7.5 to 24 × 21 cm2. The average operation time, intraoperative blood loss and length of hospital stays were 6.73 ± 1.49 h, 280.95 ± 59.25 ml and 30.55 ± 2.52 days, respectively. Eighteen flaps were designed in a single fashion, while four were in bilobed fashion. Twenty patients underwent fasciocutaneous flap transplantations, while two underwent musculocutaneous flap transplantations. Two cases developed local lysis of the flap which healed after further debridement. Direct suture of the incision after flap harvest was performed in 16 cases, while additional full-thickness skin grafting was performed in the remaining 6 cases. Further bone transport procedures were performed in 15 patients who had severe tibia bone defects. The blood flow of donor posterior tibial artery was confirmed in all patients during follow-ups. All patients recovered flap sensation at the final follow-up. The postoperative follow-ups ranged from 18 to 84 months, and no long-term complications were observed. Conclusions The modified donor blood flow-preserved cross-leg anterolateral thigh flap procedure is an ideal method to repair severe lower limb trauma with tibial artery occlusion which avoids sacrificing the major artery of the uninjured lower limb.

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